Fungal immunogens and related materials and methods

ABSTRACT

The present disclosure provides immunogenic materials and methods useful for reducing the risk of fungal infections, particularly valley fever. The disclosure also provides assays for identifying compounds useful to treat valley fever, as well as methods for ameliorating the symptoms of valley fever.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/841,921 filedDec. 14, 2017, now allowed, is a continuation of U.S. Ser. No.14/774,504 filed Sep. 10, 2015, U.S. Pat. No. 9,884,097 issued Feb. 6,2018, which claims priority to PCT No. PCT/US2014/023606 filed Mar. 11,2014, which claims the benefit of U.S. Ser. No. 61/776,770 filed Mar.11, 2013, and U.S. Ser. No. 61/777,845 filed Mar. 12, 2013, thedisclosures of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. P01AI061310 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application is being filed electronically via the USPTO EFS-WEBserver, as authorized and set forth in MPEP§ 1730 II.B.2(a)(A), and thiselectronic filing includes an electronically submitted sequence (SEQ ID)listing. The entire content of this sequence listing is hereinincorporated by reference for all purposes. The Sequence Listing, filedelectronically and identified as 3726_54785_SEQ_LIST_UA12-127, wascreated on Mar. 7, 2014, is 34,266 kb in size.

Sequence Listing Brief Descriptions SEQ ID NO Brief Description 1Reference CPS1 gene product; protein; amino acid sequence 2 ReferenceCPS1 genomic DNA; nucleic acid sequence 3-10 Primers

BACKGROUND OF INVENTION

Coccidioides species (C. immitis and C. posadasii) are the causativeagents of coccidioidomycosis (Valley Fever), an important emergingdisease endemic to the southwestern US as well as parts of Mexico andcentral and South America. Infection begins with inhalation ofarthroconidia that initiate the parasitic phase in lungs and can resultin a respiratory infection or if not controlled, a more seriousdisseminated disease.

Coccidioides spp. are dimorphic and produce a unique parasitic phasestructure, the spherule, via a switch from polar to isotrophic growthwith the spherule expanding from a barrel-shaped arthrocondium that is3-6 μm by 2-4 μm in size to a sphere 80-100 microns in diameter.Internal septation and spore formation results in production of hundredsof endospores that if released can disseminate and reinitiate spheruleformation at other places in the body. Most infections are mild andresolve without medical intervention although about 30% of infectionscause flu-like symptoms that may take 1-4 months to resolve.

A variety of approaches have been used to understand genes important forthe parasitic phase of these and other fungi. These include randommutagenesis, targeted disruption of parasitic phase-specific genes andtargeted mutagenesis of genes identified in other pathogens as virulencefactors. In addition, expression analyses have been performed toidentify phase-specific or phase-induced genes. For Coccidioides, bothexpression analyses and the latter two mutagenesis approaches have beenused, resulting in a number of mutants, some of which are altered invirulence. For example, SOWgp and MEP1 have been knocked out and theresulting mutant strains are reduced in virulence.

Over the last 50 years, many approaches to vaccination againstcoccidioidomycosis have been tried, including whole killed cells, livemutant vaccines that have been modified in virulence, partially purifiedcellular extracts, and recombinant proteins that were identified by amyriad of both low and high technology methods. To date, killed wholecell vaccines provide the best protection in mice but are nottransferable to humans because of intolerable adverse effects and poorefficacy. Recombinant proteins offer the safest approach but have modestefficacy in mice and have not been tried in a higher species.

SUMMARY OF THE INVENTION

Without being held to any particular theory, the inventors havediscovered a gene in fungi, which, if disrupted, results in leaky orotherwise more vigorous immune response-provoking variations on the wildtype fungus. Further, these cyclic peptide synthase Cps1 analog (CPS1),“CPS1 analog” deletion mutant fungal spores are not virulent;introduction of the immunogens will not result in immunogen-inducedpathology in an animal exposed to the immunogen.

The inventors demonstrate herein that CPS1 is essential for virulence inthe mouse model of coccidioidomycosis and that pre-infection of micewith a CPS1 mutant strain protects mice against subsequent infectionwith wild type Coccidioides.

The present disclosure therefore provides compositions comprising afungus having a dysfunctional CPS1 gene product, wherein the compositionis avirulent and capable of inducing an immune response in a mammal.

Also provided are such compositions, wherein the dysfunctional CPS1 geneproduct is a result of a deletion of at least a portion of the CPS1gene.

Also provided are such compositions, wherein the dysfunctional CPS1 geneproduct is a result of a deletion in a region of the CPS1 gene selectedfrom the group consisting of: at least about the entire CPS1 gene; atleast about the entire DMAP region of the CPS1 gene; at least about anentire AMP binding domain region of the CPS1 gene; a regulatory elementof the CPS1 gene; at least the coding sequence of the CPS1 gene; atleast about 90% of the CPS1 gene; at least about 80% of the CPS1 gene;at least about 70% of the CPS1 gene; at least about 60% of the CPS1gene; at least about 50% of the CPS1 gene; at least about 40% of theCPS1 gene; at least about 30% of the CPS1 gene; at least about 20% ofthe CPS1 gene; at least about 10% of the CPS1 gene.

Also provided are such compositions, wherein the dysfunctional CPS1 geneproduct is a result of deletion of the entire CPS1 gene.

Also provided are such compositions, wherein the composition is capableof inducing an immune response as a result of secretion of a metaboliteor small molecule.

Also provided are such compositions, wherein the composition is capableof inducing an immune response selected from the group consisting of:neutrophil invasion; granuloma formation; resistance to mycosis; andimmunity to mycosis.

Also provided are such compositions, wherein the fungus is aCoccidioides spp.

Also provided are such compositions, wherein the composition is capableof inducing resistance to coccidioidomycosis (valley fever).

Also provided are such compositions, wherein the composition is capableof inducing immunity to coccidioidomycosis (valley fever).

Also provided are such compositions, wherein the fungal cell is selectedfrom the group consisting of: Coccidioides immitis; Coccidioidesposadasii; Aspergillus fumigatus; Aspergillus flavus; Histoplasmacapsulatum; Blastomyces dermatitidis; Cryptococcus neoformans;Cryptococcus laurentii and Cryptococcus albidus; Cryptococcus gattii;Candida albicans; Candida glabrata; Saccharomyces boulardii; Candidatropicalis; Candida krusei; and Candida parapsilosis.

Also provided are such compositions, wherein the fungal cell isMagnaporthe oryzae.

Also provided are such compositions, which are formulated as a vaccine.

Also provided are such compositions, wherein the composition comprisesfurther avirulence protection means.

Also provided are such compositions, wherein the further avirulenceprotection means is selected from the group consisting of; amino acidbiosynthesis knockout; truncation; aging; modification; killing;formulation; resistance to reversion to wild type; and fusion.

Also provided are such compositions, which is a mammalian immunogen.

Also provided are such compositions, which is a human immunogen.

The present disclosure also provides methods of preparing apharmaceutical composition for passive immunization of an individual inneed of immunization comprising: mixing a composition herein with asuitable excipient or carrier, thereby forming a pharmaceuticalcomposition.

The present invention also provides methods of eliciting an immuneresponse in a mammal comprising: administering to a mammal apharmaceutically-effective dose of a composition herein.

Also provided are such methods wherein the composition is administeredby injection.

Also provided are such methods wherein the composition is administeredintranasally.

Also provided are such methods wherein the pharmaceutical composition isformulated for subcutaneous, intramuscular, and/or intraperitonealadministration.

Also provided are such methods wherein the pharmaceutical composition isformulated for intranasal administration.

Also provided are such methods wherein the fungal virulence isattenuated or eliminated in any mammal susceptible to the fungus.

Also provided are such methods wherein the mammalian subject is selectedfrom the group consisting of: laboratory animal; companion animal; draftanimal; meat animal; zoo animal; and human.

Also provided are such methods wherein the subject is a mammal selectedfrom the group consisting of: cat; dog; horse; bovine; camelids; andhuman.

Also provided are such methods wherein the dose of compositionadministered is selected from the group consisting of: at least about500 spores of the composition; at least about 1,000 spores of thecomposition; at least about 10,000 spores of the composition; at leastabout 20,000 spores of the composition; at least about 30,000 spores ofthe composition; at least about 40,000 spores of the composition; atleast about 50,000 spores of the composition; at least about 60,000spores of the composition; at least about 70,000 spores of thecomposition; at least about 80,000 spores of the composition; at leastabout 90,000 spores of the composition; at least about 100,000 spores ofthe composition; at least about 150,000 spores of the composition; atleast about 200,000 spores of the composition; at least about 300,000spores; at least about 500,000 spores.

Also provided are such methods which further comprise administering atleast a second subsequent dose of the composition to the mammal.

Also provided are such methods wherein the at least second subsequentdose is administered at a time interval selected from the groupconsisting of: approximately one week after the first dose;approximately two weeks after the first dose; approximately three weeksafter the first dose; approximately four weeks after the first dose;approximately five weeks after the first dose; approximately six weeksafter the first dose; approximately seven weeks after the first dose;and approximately eight weeks after the first dose.

The present disclosure also provides methods to reduce the pathogeniceffects of Coccidioides, comprising administering a siRNA complementaryto CPS1 mRNA transcripts.

The present disclosure also provides methods for screening compoundsuseful to treat coccidioidomycosis, comprising expressing CPS1 in a testmodel, introducing a test compound to the test model, and identifyingthose compounds which disrupt the function of CPS1 gene product asuseful to treat coccidiodiodomycosis.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the Patent Office upon request and payment of thenecessary fee.

FIG. 1. C57BL/6 mice challenged with intranasal doses of Δcps1 strainranging from 50-4400 spores. All survived infection for 28 days, while100% of mice challenged with WT C. posadasii succumbed by day 18(p<0.01). Closed circles, open circles, closed diamonds, and opendiamonds are CPS1 deletion mutants (Δcps1) with spore doses of 50, 211,810, and 4400, respectively; open triangles: WT, 59 spores.

FIG. 2. Protection of C57BL/6 mice by live vaccination with Δcps1strain. Quantitative total lung colony forming units (CFU) 14 daysfollowing challenge with 90 spores of WT C. posadasii were significantlyreduced in mice vaccinated with ΔCPS1 strain intraperitoneally (Δcps1IP) or subcutaneously (Δcps1 SC) compared to the positive controlchimeric Ag (Chim Ag) or adjuvant alone (Adj only) (P=0.001).

FIGS. 3A-3B. Spherule morphology comparison: NSG and C57 mice. FIG. 3A.NSG mice (NOD-SCID—No lymphocytic origin cells, no NK cells) werechallenged with 10,000 spores intranasally (IN) of Δcps1 and sacrificedfor histopathology. Spherules are occasional, thin-walled, andirregularly shaped. FIG. 3B. WT spherules in C57BL/6 mice afterinfection with 50 spores showing thick walls and very round shape ofnormal spherules.

FIGS. 4A-4D. Spherule morphology comparison between WT and Δcps1strains: FIG. 4A. WT Silveira C. posadasii strain, day 3, C57BL/6. FIGS.4B-4-D. Δcps1 C. posadasii-strain day 3.

FIGS. 5A-5B. Spherule morphology comparison: BALB/c mice with Δcps1strain. FIG. 5A. 10× imagine of very few spherules observed in NSG mice.FIG. 5B. 40× image of the area represented by the circle in A.

FIG. 6. Effects of vaccination of BALB/c mice with attenuated Δcps1strain on wild type infection. Proportion of mice surviving followinginjection with wild type Silveira strain following vaccination withΔcps1 strains intranasally (circles), Δcps1 strain injectedsubcutaneously (triangle), positive control chimeric Ag (squares) oradjuvant alone (diamonds).

FIG. 7. Lung fungal burden of vaccinated BALB/c mice surviving 28 daysafter infection. Total lung colony forming units (CFU) was measured insurviving mice 28 days after infection with 46 spores of the wild typestrain Silveira. Group 1 represents those that received intranasalvaccination with Δcps1 strain. Group 2 represents those that receivedsubcutaneous vaccination with Δcps1 strain.

FIG. 8. Coccidioides-specific staining showing wild type strain spheruleformation. Characterized by thick walls with well-developed endosporesat day 4; very little host inflammatory reaction surrounding it.Swiss-Webster mouse, high magnification.

FIGS. 9A-9B. Coccidioides-specific staining showing variable sizedspherules following Δcps1 inoculation. Two NOD-SCID and two BALB/c micewere infected intranasally with 10,000 spores of Δcps1. The sectionswere stained specifically for Coccidioides at day 3 following infection.FIG. 9A. Low magnification view. FIG. 9B. Larger magnification view

FIG. 10. H&E staining of Δcps1 spherule on day 3. High magnificationshows collapsing and degenerating spherule wall of the mutant strainwith abundant host inflammation surrounding it and inside it.

FIG. 11. H&E staining of Δcps1 spherules following rupture between day 4and 5. Lung sections between day 4 and 5 were harvested and stained withH&E to show neutrophils around and within the degenerating spherules atlow magnification. The majority of these are dead.

FIG. 12. Δcps1 spherule day 5 post-infection. The endospores are heavilysurrounded by host neutrophils and are not dispersing or enlarging tomake new spherules. Coccidioides-specific stain, 40× magnification.

FIGS. 13A-13B. Spherules at 10 days following infection with Δcps1. 10days following infection lung sections were taken and stained to showscattered granulomas with fewer than a dozen empty spherules within.FIG. 13A. H/E staining, low magnification FIG. 13B.Coccidioides-specific staining, higher magnification.

FIG. 14. Lung fungal burden following IP and SC vaccinations of Δcps1spores, Δryp1 spores, and controls. Box plot of lung fungal burden frommice vaccinated with Δcps1 or Δryp1 spores, either IP or SC, compared tocontrols.

DETAILED DESCRIPTION OF THE INVENTION

The inventors designed and characterized a Coccidioides cyclic peptidesynthase Cps1 (herein referred to as CPS1) mutant and determined itsusefulness as a potential immune response-provoking agent for protectionagainst coccidioidomycosis (Valley fever).

The inventors constructed a targeted gene-replacement strain of C.posadasii strain Silveira deleting the gene CIMG_03303.3 (misannotatedas CPSG_02657.2 and CPSG_02658.2 in Silveira) usingAgrobacterium-mediated transformation. This gene encodes an 1879 aminoacid protein that exhibits 78% similarity to C. heterostrophus CPS1 andcontains two conserved AMP-binding domains and an N-terminal DMAP1binding domain, herein referred to as cyclic peptide synthase Cps1(CPS1).

The hyphal growth rate of the deletion mutant CPS1 (Δcps1) strain wassomewhat reduced compared to Silveira at 24° C., but there was nosignificant difference at 37° C. In vitro analysis of the parasiticphase indicated that the Δcps1 mutant is able to form spherules althoughthey are reduced in size relative to Silveira.

Surprisingly, the inventors discovered that when Δcps1 arthroconidiawere introduced into susceptible C57BL/6 mice via intranasalinoculation, no disease occurred, demonstrating it is avirulent and thatCps1 protein is a virulence factor in Coccidioides. For C57BL/6 mice, 50arthroconidia are a lethal dose, while for Δcps1 strains, even when micewere inoculated with 5000 spores, no signs of disease were observed; andall mice remained healthy. In only one of the 20 mice inoculatedwith >800 Δcps1 spores, was any Coccidioides recovered, a single colonyfrom the lung of that mouse. The inventors herein describe that Δcps1fungi can induce protection against further infection and act as a livevaccine.

As exemplified herein, the inventors injected C57BL/6 mice eitherintraperitoneal or subcutaneously with 50,000 Δcps1 spores and boostedwith the same amount after two weeks. Four weeks later, the mice werechallenged intranasally with 90 spores of Coccidioides strain Silveira.

The control mice all developed disease, but the inventors show that theΔcps1 mutant provides complete protection against infection, with nosign of disease in infected mouse lungs or other organs.

Interestingly, at the site of injection of Δcps1 arthroconidia, all micehad evidence of granulomatous lesions, indicating local reproduction ofthe mutant. The present disclosure therefore provides, inter alia,mutant strains of fungus as live vaccines against fungal pathologies,including coccidioidomycosis.

Further, via metabolite analysis of WT and the Δcps1 mutant, theinventors have observed small molecule differences between the strains.

The small metabolite product and the gene are targets for drugtherapeutics. Because this gene is conserved among fungi, it is ageneral target for therapeutics in treatment of fungal diseases.

Previous efforts at development of fungal vaccines were based on eitherwhole cell extracts, partially purified extracts or recombinantproteins. None of the previous attempts produced the protection seenwith the present mutant strains.

Unlike other live attenuated vaccines, Cps1 may be part of an enzymecomplex that produces one or more small molecules that may have a rolein virulence. The invention of the CPS1 mutant could lead to a vaccineor to targets for treatment either through binding of the smallmolecules involved in virulence or by binding or disrupting the proteininvolved in making the small molecules required for virulence.

Definitions

Coccidioides cyclic peptide synthase Cps1, herein referred to as Cps1,is a protein encoded by a CPS1 gene product and CPS1 mRNA, and mayencode either wild type or a mutant. A wild type or mutant CPS1 geneproduct will encode for a Cps1 protein.

The term “dysfunctional,” “non-functional,” “inactivated,” or“inactivation” when referring to a gene or a protein means that theknown normal function or activity of the gene or protein has beeneliminated or highly diminished. For example, inactivation of CPS1 canbe effected by inactivating the CPS1 gene. Inactivation which rendersthe gene or protein dysfunctional includes such methods as deletions,mutations, substitutions, interruptions or insertions in the nucleicacid gene sequence.

General techniques of genetic recombination, including vectorconstruction, transformation, selection of transformants, host cellexpression, etc., are further described in Maniatis et al, 1989,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; Ausubel et al., 1989, Current Protocolsin Molecular Biology, Greene Publishing Associates & Wiley Interscience,N.Y.; Innis et al. (eds), 1995, PCR Strategies, Academic Press, Inc.,San Diego, Calif.; and Erlich (ed), 1992, PCR Technology, OxfordUniversity Press, New York. Agrobacterium transformation and replacementof Coccidioides genes was as described in Abuodeh et al. 2000, GeneticTransformation of Coccidioides immitis facilitated by Agrobacteriumtumefaciens. Journal of Infectious Diseases, 181:2106-2110 and Kellneret al., 2005, Coccidioides posadasii contains a single 1,3-beta-glucansynthase gene that appears to be essential for growth, Eukaryotic Cell,4:111-120.

As used herein, a “therapeutically effective amount” means a quantity ofa specified pharmaceutical or therapeutic compound or compositionsufficient to achieve a desired effect in a subject, or in a cell, beingtreated with the compound or composition. The effective amount of theagent will be dependent on several factors, including, but not limitedto, the subject or cells being treated, and the manner of administrationof the therapeutic composition.

As is conventional in the art, the term “attenuated” refers to a cell,culture, or strain of fungus (e.g. Coccidioides spp.) exhibiting adetectable reduction in infectivity or virulence in vitro and/or in vivoas compared to that of the parent strain of the fungus from which theattenuated cell, culture, or strain is derived. Reduction in virulenceencompasses any detectable decrease in any attribute of virulence,including infectivity in vitro and/or in vivo, or any decrease in theseverity or rate of progression of any clinical symptom or conditionassociated with infection.

The term “avirulent”, as used herein, does not mean that a microbe ofthat genus or species cannot ever function as a pathogen, but that theparticular microbe being used is avirulent with respect to theparticular animal being treated. The microbe may belong to a genus oreven a species that is normally pathogenic but must belong to a strainthat is avirulent. The microbe may also be modified genetically orthrough avirulence protection means to make the microbe avirulent.Examples of avirulent means include, but are not limited to, geneticengineering to knock out genes required for virulence, amino acidbiosynthesis knockout, truncation of the viral genome, aging, killing,formulation, resistance to reversion to wild type, and fusion.“Pathogenic,” as used herein, means capable of causing disease orimpairing normal physiological functioning. An “avirulent strain” isincapable of inducing the full set of symptoms of the disease that isnormally associated with its virulent pathogenic counterpart. The term“microbes,” as used herein, includes bacteria, protozoa, and fungi.Derivatives of avirulent Coccidioides spp. are also contemplated to bewithin the scope of this disclosure. By “derivative” it is meantsexually or asexually derived progeny and mutants of the avirulentstrains including single or multiple base substitutions, deletions,insertions or inversions which retain the inability to producefunctional Cps1 protein. For example, the Coccidioides posadasiiSilveira strain that has a deletion of the CPS1 gene described herein.

The term “immunogen,” “immunogens,” “antigen,” or “antigens” means amaterial that can induce an immune response and is therefore antigenic.By “immune response” means any reaction by the immune system. Thesereactions include the alteration in the activity of an organism's immunesystem in response to an antigen and may involve, for example, antibodyproduction, induction of cell-mediated immunity, complement activationor development of immunological tolerance. Immune response to antigensis well studied and widely reported. A survey of immunology is given inBarrett, James, T., Textbook of Immunology: Fourth Edition, C.V. MosbyCo., St. Louis, Mo. (1983). More specifically, the present disclosureprovides a live, attenuated fungus (e.g. Coccidioides spp.) that can beused as an immunogenic composition or a vaccine. It will be appreciatedthat the attenuated fungus contains a dysfunctional CPS1 gene.

“Vaccine,” as used herein, means an agent used to stimulate the immunesystem of a living organism so that protection against future harm isprovided. “Immunization” refers to the process of inducing a continuinghigh level of antibody and/or cellular immune response in whichT-lymphocytes can either kill a pathogen and/or activate other cells(e.g., phagocytes) to do so in an organism, which is directed against apathogen or antigen to which the organism has been previously exposed.

The term “adjuvant” is intended to mean a composition with the abilityto enhance an immune response to an antigen generally by being deliveredwith the antigen at or near the site of the antigen. Ability to increasean immune response is manifested by an increase in immune mediatedprotection. Enhancement of humoral immunity can be determined by, forexample, an increase in the titer of antibody raised to the antigen.Enhancement of cellular immunity can be measured by, for example, apositive skin test, cytotoxic T-cell assay, ELISPOT assay for IFN-gammaor IL-2. Adjuvants are well known in the art. Exemplary adjuvantsinclude, for example, Freud's complete adjuvant, Freud's incompleteadjuvant, aluminum adjuvants, MF59 and QS21.

As used herein, “inhibit,” “inhibiting,” or “inhibition” includes anymeasurable or reproducible reduction in the infectivity of a fungus inthe subject. “Reduction in infectivity” means the ability of the subjectto prevent or limit the spread of the fungus in tissues or organsexposed to or infected by the fungus. Furthermore, “amelioration,”“protection,” “prevention,” and “treatment” mean any measurable orreproducible reduction, prevention, or removal of any of the symptomsassociated with fungal infectivity, and particularly, the prevention, oramelioration of infection and resultant pathology itself.

As used herein, “subject” means a patient or individual having symptomsof, or at risk for, fungal infection, coccidioidomycosis, or othermalignancy. A subject may be human or non-human and may include, forexample, laboratory animal, companion animal; draft animal, meat animal,zoo animal, and human. The subjects may include either adults orjuveniles (e.g., children). Moreover, subject may mean any livingorganism, preferably a mammal (e.g., human or non-human) that maybenefit from the administration of compositions contemplated herein.

Compositions

The current disclosure provides compositions comprising a fungus havinga dysfunctional CPS1 gene product. The compositions may be formulated asan ingredient in a pharmaceutical composition, and this formulation canaid in administration of the composition. The compositions may routinelycontain pharmaceutically acceptable concentrations of salts, bufferingagents, preservatives and various compatible carriers or diluents. Forall forms of delivery, the vectors may be formulated in a physiologicalsalt solution. In one embodiment, the composition is a vaccine.

The preferred formulations of the composition may depend on the methodof administration of the composition. It is contemplated that thecomposition will include one or more conventional pharmaceuticallyacceptable carriers, adjuvants, other immune-response enhancers, and/orvehicles (collectively referred to as “excipients”). Such excipients aregenerally selected to be compatible with the active ingredient(s) in thecomposition. Use of excipients is generally known to those skilled inthe art. Suitable pharmaceutical carriers, excipients, adjuvants and thepreparation of dosage forms are described in, for example, Remington'sPharmaceutical Sciences, 17th Edition, (Gennaro, Ed., Mack PublishingCo., Easton, Pa., 1985.

Mucosal compositions may be, for example, liquid dosage forms, such aspharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs. Excipients suitable for such vaccine preparations include,for example, inert diluents commonly used in the art, such as, water,saline, dextrose, glycerol, lactose, sucrose, starch powder, celluloseesters of alkanoic acids, cellulose alkyl esters, talc, stearic acid,magnesium stearate, magnesium oxide, sodium and calcium salts ofphosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol. Excipients also cancomprise various wetting, emulsifying, suspending, flavoring (e.g.,sweetening), and/or perfuming agents.

The compositions may be formulated for intranasal administration with apharmaceutically acceptable carrier such as water, buffered saline,ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like) suitable mixtures thereof, orvegetable oils. If necessary, the action of contaminating microorganismsmay be prevented by various antibacterial agents, such as parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. It willoften be preferable to include in the formulation isotonic agents, forexample, glucose or sodium chloride. Such formulation may beadministered intranasally as an aerosol or atomized spray, or as liquiddrops.

Oral mucosal compositions also may, for example, be tableted orencapsulated for convenient administration. Such capsules or tablets cancontain a controlled-release formulation. In the case of capsules,tablets, and pills, the dosage forms also can comprise buffering agents,such as sodium citrate, or magnesium or calcium carbonate orbicarbonate. Tablets and pills additionally can be prepared with entericcoatings.

“Parenteral administration” includes subcutaneous injections, submucosalinjections, intravenous injections, intramuscular injections,intrasternal injections, and infusion. Injectable preparations (e.g.,sterile injectable aqueous or oleaginous suspensions) can be formulatedaccording to the known art using suitable excipients, such as vehicles,solvents, dispersing, wetting agents, emulsifying agents, and/orsuspending agents. These typically include, for example, water, saline,dextrose, glycerol, ethanol, corn oil, cottonseed oil, peanut oil,sesame oil, benzyl alcohol, benzyl alcohol, 1, 3-butanediol, Ringer'ssolution, isotonic sodium chloride solution, bland fixed oils (e.g.,synthetic mono- or diglycerides), fatty acids (e.g., oleic acid),dimethyl acetamide, surfactants (e.g., ionic and non-ionic detergents),propylene glycol, dextran, lactose, trehalose, and/or polyethyleneglycols. Excipients also may include small amounts of other auxiliarysubstances, such as pH buffering agents.

The compositions may include one or more adjuvants that enhance asubject's immune response (which may include an antibody response,cellular response, or both), thereby increasing the effectiveness as avaccine. The adjuvant (s) may be a substance that has a direct (e.g.,cytokine or Bacille Calmette-Guerin (BCG)) or indirect effect(liposomes) on cells of the subject's immune system. Examples of oftensuitable adjuvants include oils (e.g., mineral oils), metallic salts(e.g., aluminum hydroxide or aluminum phosphate), bacterial components(e.g., bacterial liposaccharides, Freund's adjuvants, and/or MDP), plantcomponents (e.g., Quil A), cytokines and/or one or more substances thathave a carrier effect (e.g., bentonite, latex particles, liposomes,and/or Quil A). Adjuvants also include, for example, CARBIGEN adjuvantand carbopol. It should be recognized that this disclosure encompassesboth compositions that include an adjuvant (s), as well as compositionsthat do not include any adjuvant.

“Cytokines” used in the compositions and methods described herein, referto small proteins secreted primarily, but not exclusively, by cells ofthe immune system that promote the proliferation and/or differentiativefunctions of other cells. Examples of cytokines include interleukins,interferons, hematopoietic colony stimulating factors (CSF), andproinflammatory factors such as tumor necrosis factor (TNF). It iscontemplated that the compositions may be freeze-dried (or otherwisereduced in liquid volume) for storage, and then reconstituted in aliquid before or at the time of administration. Such reconstitution maybe achieved using, for example, vaccine-grade water.

Administration of the Compositions

In accordance with particular embodiments, the composition comprisingfungi having a dysfunctional CPS1 gene product is used in a vaccinepreparation. In general, the vaccine is administered in animmunologically effective amount, which is an amount sufficient toinduce a protective immune response in the subject against fungalinfection (e.g. Coccidioides spp.). The live attenuated fungi describedherein are capable of triggering an immune response that protects amammal against fungal infection or colonization after one or moreadministrations as a live vaccine. A “protective immune response” refersto any immunological response, either antibody or cell-mediatedimmunity, or both, occurring in the subject that either prevents ordetectably reduces subsequent infection, or eliminates or detectablyreduces the severity, or detectably slows the rate of progression, ofone or more clinical symptoms or conditions associated with fungalinfection.

The immunogenicity level may be determined experimentally by challengedose titration study techniques generally known in the art. Suchtechniques typically include vaccinating a number of subjects with thevaccine at different dosages, and then challenging the subjects with thevirulent fungi to determine the minimum protective dose.

Factors affecting the preferred dosage regimen may include, for example,the age, weight, sex, diet, activity, and condition of the subject; theroute of administration; the efficacy, safety, and duration-of-immunityprofiles of the particular vaccine used; whether a delivery system isused; and whether the vaccine is administered as part of a drug and/orvaccine combination. Thus, the dosage actually employed can vary.Determining such dosage adjustments is generally within the skill ofthose in the art using conventional means.

In one embodiment, the composition is administered at a dose of at leastabout 500 spores of the composition, at least about 1,000 spores of thecomposition, at least about 10,000 spores of the composition, at leastabout 20,000 spores of the composition, at least about 30,000 spores ofthe composition, at least about 40,000 spores of the composition, atleast about 50,000 spores of the composition, at least about 60,000spores of the composition, at least about 70,000 spores of thecomposition, at least about 80,000 spores of the composition, at leastabout 90,000 spores of the composition, at least about 100,000 spores ofthe composition, at least about 150,000 spores of the composition, andat least about 200,000 spores of the composition.

It is contemplated that the compositions may be administered to asubject at a single time; or, alternatively, two or more times overdays, weeks, months, or years. In some embodiments, the composition isadministered at least two times. In some such embodiments, for example,the compositions are administered twice, with the second dose (e.g., thebooster) being administered approximately one week after the first dose,approximately two weeks after the first dose, approximately three weeksafter the first dose, approximately four weeks after the first dose,approximately five weeks after the first dose, approximately six weeksafter the first dose, approximately seven weeks after the first dose,and approximately eight weeks after the first dose. In the aboveembodiments, the first and subsequent dosages may vary, such as, forexample, in amount and/or form. Often, however, the dosages are the sameas to amount and form.

In certain embodiments, the compositions are administered to a subjectthat is immunogenically naive to the fungi, e.g., the subject has notbeen vaccinated for the fungus or exposed to the fungus. In accordancewith this embodiment, the composition is administered before the subjectrecipient is infected with the fungus. In such embodiments, the vaccinepreparation may, for example, be administered to prevent, reduce therisk of, or delay the onset of Coccidioides spp. infection or one ormore (typically two or more) Coccidioides spp. symptoms.

In some embodiments, the compositions are administered to subjects in apopulation after a subject in the population has been infected with thefungus. In such embodiments, the compositions may, for example,ameliorate, suppress, or eradicate the fungus or one or more (typicallytwo or more) fungal symptoms in the subjects of the population.

The compositions can be administered by conventional means, including,for example, mucosal administration, (such as intranasal, oral,intratracheal, and ocular), and parenteral administration (such as,without limitation, intraperitoneal, subcutaneous or intramuscularadministration). The compositions may also be administered intradermallyor transdermally (including, without limitation, via a skin patch ortopical administration). Mucosal administration is often particularlyadvantageous for live attenuated vaccines.

Cyclic Peptide Synthase Cps1 (CPS1)

CPS1 gene was originally identified as a potential non-ribosomal peptidesynthase component, because it encodes an 1879 amino acid polypeptidewith two AMP binding domains related to the adenylation domains inbacterial non-ribosomal peptide synthases. However it also contains aputative N-terminal DMAP1b domain. In mammals, this domain binds theDMAP1 transcriptional co-repressor that has been shown to bindregulatory proteins and is proposed to act as a co-repressor oftranscription.

The present CPS1 deletion strains are non-pathogenic but do initiate theformation of spherules (the infectious form of Coccidioides). Themutants also form spherules in vitro. The CPS1 mutant appears to havegreat potential as an attenuated vaccine since it protects frominfection. When susceptible mice are challenged with wild type C.posadasii after inoculation with the Δcps1 mutant strain, nearly allexperience extended survival of at least four weeks and have low fungalburdens. Inoculation with the CPS1 deletion mutant results in animalsthat are completely resistant to infection. Other fungi can begenetically engineered to make a fungal strain having a dysfunctionalCPS1 gene to make vaccines useful to treat a number of different fungalinfections.

In some embodiments, the dysfunctional CPS1 gene is a result of adeletion of at least a portion of the CPS1 gene. In certain embodiments,the dysfunctional CPS1 gene product is a result of a deletion of atleast about the entire CPS1 gene, at least about the entire DMAP regionof the CPS1 gene, at least about an entire AMP binding domain region ofthe CPS1 gene, a regulatory element of the CPS1 gene, at least thecoding sequence of the CPS1 gene, at least about 90% of the CPS1 gene,at least about 80% of the CPS1 gene, at least about 70% of the CPS1gene, at least about 60% of the CPS1 gene, at least about 50% of theCPS1 gene, at least about 40% of the CPS1 gene, at least about 30% ofthe CPS1 gene, at least about 20% of the CPS1 gene, and at least about10% of the CPS1 gene. In another embodiment, the dysfunctional CPS1 geneproduct is a result of deletion of the entire CPS1 gene.

Coccidioidomycosis (Valley Fever)

Coccidioidomycosis is a fungal infection (Valley Fever) caused by theendemic fungal species, Coccidioides immitis and Coccidioides posadasii.The disease can range from an asymptomatic infection that renders humansimmune for life to a fatal respiratory or disseminated infection.Approximately 40% of 150,000 people infected annually become sick, andapproximately 5% develop severe and life-threatening illness that mayleave them disabled, under continuous treatment, or deceased. Amongpatients that develop disseminated disease, most are treated for monthsto years, and many who discontinue treatment suffer relapse at somepoint because the current drugs available suppress but do not eradicatethe fungus from the body. Treatment with antifungal medication may costbetween $5000-$20,000 per year, not including the costs of ancillarycare such as hospitalization, rehabilitation, frequent medical care, anddisability. Better vaccines and medications to treat illness, e.g.,short morbidity and reduce the severity and life time sequelae ofcoccidioidomycosis, are desperately needed because currently availablemedications are primarily fungistatic and fail in a significant numberof disseminated cases.

Coccidioidomycosis is commonly known as cocci or “Valley Fever”, as wellas “California Fever”, “desert rheumatism”, and “San Joaquin ValleyFever”, is endemic in certain parts of Arizona, California, Nevada, NewMexico, Texas, Utah and northern Mexico.

Coccidioides immitis or Coccidioides posadasii resides in the soil incertain parts of the southwestern United States, most notably inCalifornia and Arizona. It is also prevalent in northern Mexico, andparts of Central and South America. It is dormant during long dryspells, then develops as a mold with long filaments when the rains comeand that matures to break off into spores. The spores, known asarthroconidia, are swept into the air by disruption of the soil, such asduring construction, farming, windstorms or an earthquake.

Infection is caused by inhalation of the particles. The disease is nottransmitted from person to person. The infection ordinarily resolvesleaving the patient with a specific immunity to re-infection. However,in some cases the infection may manifest itself repeatedly orpermanently over the life of the host. Coccidioides immitis orCoccidioides posadasii is a dimorphic saprophytic organism that grows asa mycelium in the soil and produces a spherule form in the hostorganism.

Coccidioidomycosis is confined to the western hemisphere between 40° Nand 40° S. Dry soil, especially in the Lower Sonoran Life Zone, issupportive of the pathogenic fungi growth. In harmony with mycelium lifecycle, incidence increases with periods of dryness after a rainy season;this phenomenon, termed “grow and blow,” refers to growth of the fungusin wet weather, producing spores which are spread by the wind duringsucceeding dry weather.

Besides humans, dogs, and cats, the fungus can be shown to infect mostmammals, even if they do not get sick from it very often. Species inwhich Valley Fever has been found include livestock such as cattle andhorses; llamas; marine mammals, including sea otter; zoo animals such asmonkeys and apes, kangaroos, tigers, etc; and wildlife endemic to thegeographic area such as cougar, skunk, and javelina.

In soil, Coccidioides spp. exists in filament form. It forms hyphae inhorizontal and vertical direction. With time, cells within hyphaedegenerate to form alternating barrel shaped cells, approximately 3-5microns in size, called arthroconidia. Arthroconidia are lightweight andcarried by air currents. They can easily be inhaled without the personknowing. On arriving in alveoli, they enlarge in size and internalseptations are developed, forming a structure termed a spherule.Internal spores, termed endospores develop within the spherule as itmatures. Rupture of the spherules release these endospores, which inturn repeat the cycle and spread the infection locally and candisseminate to any organ via the blood and lymph systems. Nodules canform in lungs surrounding these spherules. When these rupture, theyrelease their contents into bronchus, forming thin-walled cavities.These cavities can result in symptoms like characteristic chest pain,meoptysis and persistent cough.

Aspergillosis

Aspergillus spp. are fungi whose spores are present in the air webreathe, but does not normally cause illness. However an individual witha weakened immune status may be susceptible to infection by someAspergillus species, primarily Aspergillus fumigatus.

Aspergillosis is a group of diseases which can result from Aspergillusinfection and includes invasive aspergillosis, ABPA, CPA andaspergilloma. Some asthma patients with very severe asthma may also besensitized to fungi like Aspergillus (SAFS).

Aspergillosis may affect patients whose immune system may becompromised—including those with leukemia, chemotherapy patients orthose on steroids, transplant patients, cystic fibrosis, HIV or AIDS,chronic obstructive pulmonary disease (COPD), chronic granulomatousdisease (CGD), severe asthma with fungal sensitivity (SAFS) and manyothers.

Aspergillus does not solely affect humans; birds and animals can alsodevelop aspergillosis, and some plant diseases and food spoilage may bedue to Aspergillus infection. Especially serious are those Aspergillusspecies that produce the highly carcinogenic mycotoxin, aflatoxin, whichcan cause serious acute and chronic health problems in people whoaccidentally ingest it. Primarily aflatoxin producing species are A.flavus and A. parasiticus, which can contaminate foods such as maize,peanuts and cottonseeds.

Histoplasmosis

Histoplasmosis is a disease caused by the fungus Histoplasma capsulatum.The fungus lives in the environment, usually in association with largeamounts of bird or bat droppings. Lung infection can occur after aperson inhales airborne, microscopic fungal spores from the environment;however, many people who inhale the spores do not get sick. The symptomsof histoplasmosis are similar to pneumonia, and the infection cansometimes become serious if it is not treated.

Blastomycosis

Blastomycosis is a disease caused by the fungus Blastomycesdermatitidis. The fungus lives in moist soil and in association withdecomposing organic matter such as wood and leaves. Lung infection canoccur after a person inhales airborne, microscopic fungal spores fromthe environment; however, many people who inhale the spores do not getsick. The symptoms of blastomycosis are similar to flu symptoms, and theinfection can sometimes become serious if it is not treated. Blastomycesdermatitidis can also infect dogs.

Candidiasis

Candida is a yeast and the most common cause of opportunistic mycosesworldwide. It is also a frequent colonizer of human skin and mucousmembranes. Candida is a member of normal flora of skin, mouth, vagina,and stool. As well as being a pathogen and a colonizer, it is found inthe environment, particularly on leaves, flowers, water, and soil. Whilemost of the Candida spp. are mitosporic, some have known teleomorphicstate and produce sexual spores.

Infections caused by Candida spp. are in general referred to ascandidiasis. The clinical spectrum of candidiasis is extremely diverse.Almost any organ or system in the body can be affected. Candidiasis maybe superficial and local or deep-seated and disseminated. Disseminatedinfections arise from hematogenous spread from the primarily infectedlocus. Candida albicans is the most pathogenic and most commonlyencountered species among all. Its ability to adhere to host tissues,produce secretory aspartyl proteases and phospholipase enzymes, andtransform from yeast to hyphal phase are the major determinants of itspathogenicity.

Cryptococcosis

Cryptococcus neoformans is an encapsulated yeast and the causative agentof cryptococcosis. Given the neurotropic nature of the fungus, the mostcommon clinical form of cryptococcosis is meningoencephalitis. Thecourse of the infection is usually subacute or chronic. Cryptococcosismay also involve the skin, lungs, prostate gland, urinary tract, eyes,myocardium, bones, and joints. The most commonly encounteredpredisposing factor for development of cryptococcosis is AIDS. Lesscommonly, organ transplant recipients or cancer patients receivingchemotherapeutics or long-term corticosteroid treatment may developcryptococcosis.

Kits

The present disclosure further includes kits that are suitable for usein performing the methods described above. The kits can includes adosage form of the compositions in an appropriate container and can alsooptionally include at least one additional component, and, typically,instructions for using the compositions with the additionalcomponent(s). The additional component(s) may, for example, be one ormore additional ingredients (such as, for example, one or more of theexcipients discussed above) that can be mixed with the compositionsbefore or during administration. The additional component (s) mayalternatively (or additionally) include one or more apparatuses foradministering the compositions to the subject. Such an apparatus may be,for example, a syringe, inhaler, nebulizer, pipette, forceps, or anymedically acceptable delivery vehicle. In some embodiments, theapparatus is suitable for subcutaneous administration of thecompositions. In some embodiments, the apparatus is suitable forintranasal administration of the vaccine preparation.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.Whenever a range is given in the specification, all intermediate rangesand sub-ranges, as well as all individual values included in the rangesgiven are intended to be included in the disclosure. When a Markushgroup or other grouping is used herein, all individual members of thegroup and all combinations and sub-combinations possible of the groupare intended to be individually included in the disclosure.

Examples Example 1. Material and Methods

Strains, Media and Growth Conditions

Coccidioides posadasii strain Silveira (ATCC 28868) was cultured on2×GYE medium (2% glucose, 1% yeast extract and 1.5% agar) at roomtemperature. All manipulations of viable cultures were performed in theKeating Building Select agent biosafety level 3 (BSL3) laboratory, usingstandard operating procedures developed for working with this selectagent and approved by the Centers for Disease Control (CDC). Δcps1strains were selected and maintained on 2×GYE media supplemented withhygromycin at 50 μg/ml. Arthroconidia of Silveira were harvested fromfour-week old cultures grown on 2×GYE at room temperature using sterilewater by the mini-stir bar method described previously (Huppert et al.Antimicrobial Agents and Chemotherapy, 1:367-372, 1972). Arthroconidiawere washed with sterile dH₂O and stored in sterile dH₂O at 4° C. Sporecounts were made with a hemacytometer counts and viable countsdetermined by plating. Spherules were generated by growth of strains inmodified Converse medium at 37° C., 20% CO₂ and shaken at 180 rpm asdescribed in J Bacteriol., 78:231-239 (1959). Spherules were harvestedat 24-hour intervals up to 120 hours, fixed in 10% formaldehyde andstained with cotton blue. At least 50 spherules were measured toestimate their size at each time point. Statistical analysis wasperformed using SAS (version 9.1, SAS Institute Inc., Cary, N.C.) andthe size of spherule is presented as mean±SD.

Construction of CPS1 Gene Deletion Cassette

The CPS1 gene of Coccidioides, CIMG_03303.3, was identified via homologyto the Cochlibolus heterostrophus CPS1 gene. A CPS1 gene deletioncassette was constructed in multiple steps using primers listed in Table1. C. posadasii strain Silveira DNA was used as a template to generatePCR fragments representing the 5′ and 3′ flanking regions of the CPS1gene using primers OAM 1190 and OAM 1192, and OAM 1193 and OAM 1194respectively. Primers OAM 1192 and OAM 1193 contain sequencescomplementary to the ends of the hygromycin resistance gene cassette(hphB) of plasmid pCB1004. The hygromycin (hphB) gene was amplified fromplasmid pCB1004 using primers OAM597 and OAM 598. The PCR products ofthe CPS1 5′ and 3′ flanking regions were mixed with that of the hphBgene and amplified with nested primers OAM 1191 and 1195, that containEcoRI sites. The resulting PCR product was then ligated into pGEM®-TEasy (Promega, Madison, Wis.). The construct, designated pAM1567, wasverified by restriction analysis and PCR and the hphB insert gene of theplasmid was sequenced to determine that no mutations had beenintroduced. The gene replacement construct from pAM1567, containing theCPS1 5′flank-hphB-CPS1 3′flank was cloned into the binary vector pAM1145as an EcoRI fragment, producing plasmid pAM1594. Plasmid pAM1594 wastransformed into Agrobacterium tumefaciens strain EHA105 byelectroporation and the resulting strain was named as A1594.

TABLE 1 Primers Used Sense or Anti- SEQ Primer sensePrimer sequence (5′-3′) ID 5′UTR OAM1190 Sense GTGGGTATCAGTTGTTTGTAGGAAG 4 OAM1192 Anti- GCTCCTTCAATATCAGTTAACGTCGAGTTAA  5 senseACGCCAATCAGTATCGTCGTTTCG 3′ UTR Sense OAM1193 Anti-AGATGCCGACCGGGAACCAGTTAACATAGA  6 sense CATGAGGATTGCTCGGCTTTGTC OAM1194TCACGATGTCGTACGGGCCAGTTTG  7 Nested OAM1191 SenseGGGAATTCGAATTCGCGTGGTCTGGTAGTCG  8 CGTTGAGAGCC OAM1195 Anti-GAGCCGGAATTCCCTAAATGCATAGCCATTC  9 sense CACAAATAC CPS1 Intern OAM1288Sense CAACCGCAGGTCAGTGTATG 10

Targeted Disruption of the C. posadasii CPS1 Gene in Strain Silveira

C. posadasii strain Silveira was transformed using A. tumefaciens strainA1594 as described previously in Eukaryotic Cell 4:111-120 (2005).Briefly, 1×10⁷ arthroconidial germilings and 1×10⁹ A. tumefaciens cellswere mixed and dispersed onto six sterile 0.45 μM, 82 mm diameternitrocellulose filters (Millipore Corporation, Bedford, Mass., USA) onplates containing AB induction media. Following co-cultivation at roomtemperature for 48 hours, the nitrocellulose filters were transferredonto selection plates containing 2×GYE media supplemented with 50 μg/mlhygromycin (selection for transformed strains) and 100 μg/ml kanamycin(counter selection to prevent further growth of A. tumefaciens).Transformants were isolated after incubation at room temperature for 1-2weeks and grown on selection plates for sporulation. Monoconidialisolates were selected via two conidial passages as described inEukaryotic Cell 4:111-120 (2005).

DNA Isolation and Confirmation of Transformants

Genomic DNA was isolated from putative transformants as describedpreviously in Eukaryotic Cell 4:111-120 (2005). Briefly, mycelium from ayoung colony was scraped from plates and mechanically disrupted withacid-treated glass beads in lysis buffer (50 mM Tris-HCl, [pH7.5], 100mM EDTA [pH8.0], 100 mM NaCl, 0.5% SDS and 100 mM DTT) by vortexing at3000 rpm for 10 minutes. DNA was then purified by extraction withphenol:chloroform:isoamyl alcohol. Following precipitation, the DNA wastreated with RNase A and then extracted using CTAB. The DNA wasprecipitated, resuspended in 50 μl sterile dH₂O and stored at −20° C.

Transformed C. posadasii strains were analyzed by Southern blot analysisto verify replacement of the CPS1 gene by the hphB gene using standardprocedures as described previously in Eukaryotic Cell 4:111-120 (2005).Probes for hybridization included the hphB gene, generated by PCRamplification from plasmid pCB1004 using primers OAM597 and OAM 598, aninternal fragment of CPS1 generated by amplification from Silveriagenomic DNA using primers OAM1288 and OAM1289 and a 5′ flanking fragment(Table 1).

Measurement of In Vitro Growth and Spherulation

To determine whether the Δcps1 strains were altered in hyphal growth andconidiation, colonial radial growth was compared to strain Silveira.Three day-old monoconidial cultures were placed on 2×GYE petri platescontaining or lacking 50 μg/ml hygromycin. Strains were plated intriplicate and the growth rate was assessed at room temperature (24° C.)and at 37° C. Colony diameters were recorded at three-day intervals for21 days. Statistical analysis was performed using SAS (version 9.1, SASInstitute Inc., Cary, N.C.) and the data is presented as mean±SD.

Mice

8-week-old female C57/BL6 mice, were purchased fromHarlan-Sprague-Dawley (Indianapolis, Ind.) and maintained according toNational Institutes of Health guidelines. All studies were conductedwith the approval of the Institutional Animal Care and Use Committee atthe University of Arizona.

Example 2. In Vivo Studies

Pathogenicity of the Δcps1 strain was assessed in two independent mousestudies in the Animal BSL3 facility at the Department of VeterinarySciences and Microbiology at the University of Arizona. Mice wereinfected intranasally following i.p. anesthetization withketamine-xylazine as described previously in Infection and Immunity70:3287-3289 (2002). Twelve mice were inoculated per treatment with twomice sacrificed at 14 days for histopathology and the other 10maintained for 28 days or until they appeared moribund. Mice weretreated with the following spore doses (design/actual): Silveira, 50/59arthroconidia/mouse, Δcps1, 50/50, 250/211, 1000/810 spores/mouse. Inthe second study, a higher dose was used to further assess avirulence.Strain Silveira was inoculated at 50/53 arthroconidia/mouse(design/actual), while Δcps1 was infected with 4400 arthroconidia. Micewere inoculated intranasally and monitored as in the first study.

An additional mouse study was performed to test whether the Δcps1 straincould provide protection against subsequent inoculation with wild typeC. posadasii. In this study, groups of eight mice were inoculated twice,two weeks apart, with 50,000 spores of the Δcps1 strain introducedeither subcutaneously or intraperitoneally. They were then challengedintranasally four weeks after the second (booster) dose with 90 sporesof wild type parental C. posadasii, strain Silveira. As controls, eightmice were vaccinated with a positive control chimeric antigen,Ag2/_(PRAa1-106) fused to CSA, and eight were vaccinated with adjuvantalone, a combination of MPL-SE (25 μg/mouse) and CpG (10 μg/mouse). Thelungs of mice were weighed and quantitatively cultured two weeks afterchallenge.

Mice in the control group had mean lung fungal burdens of 5.3×10⁷(800-11.9×10⁷), while the mean fungal burden of all the mice vaccinatedwith CPS1 knockout was 212 cfu (range 1-1650). This avirulent strainafforded a high level of protection in a susceptible mouse, whilecausing no evidence of disease in 48 mice challenged with doses up 4400spores intranasally.

Example 3. Identification of the Coccidioides Posadasii CPS1 Gene

The CPS1 gene of Coccidioides spp. was identified using the C.heterostrophus sequence as a query (AF332878). The C. immitis strain RSgene CIMG_03303.3 encodes a protein of 1879 amino acids with a predictedmolecular mass of 208 kDa. Alignment with the C. heterostrophus proteinencoded by AF332878, indicated C. immitis CPS1 exhibited 55% identityand 13% similarity. The inventors note that the annotation for AF332878was determined to represent a truncated form of the C. heterostrophusCPS1 ORF, lacking the N-terminal end of the protein which was determinedbased on RT-PCR (B.G. Turgeon, personal communication). Thus there isgreater overall similarity between these proteins. Using BLASTX withCIMG_03303.3 against the C. posadasii Silveira genome identified twoadjacent genes CPSG_02657.2 and CPSG_02658.2 that align consecutivelywith the RS CPS1 gene. Further alignment and analysis of these proteinsequences with CIMG_03303.3 suggest an annotation error in Silveira;combining these proteins formed a complete open reading frame encodingthe CPS1 protein. CIMG_03303.3 from Coccidioides immitis strain RSshowed 99% identity to a C. posadasii strain C735 protein (C. posadasiistrain C735 delta SOWgp, XP_003068865) and 83% identity with UREG_07134(Uncinocarpus reesii 1704). The degree of similarity to Paracoccidioidesbrasiliensis ranged from 88 to 92% between the strains. The Cps1 proteinis conserved in most ascomycete fungi with varying degrees of similarityto the query sequence and is related to proteins found in animalsincluding the Dip2 proteins identified in Drosophila and found inmammals.

C. immitis Cps1 contains a DMAP1 binding domain at the N-terminus andtwo acyl-CoA synthetase (AMP-forming)/AMP-acid ligases II (CaiC)domains. The DMAP1 is known to act as a transcriptional co-repressor andthe CaiC domains were found to function in lipid metabolism andsecondary metabolite biosynthesis, transport and catabolism.

Example 4. Construction of a CPS1 Deletion Mutant

To understand the effect of CPS1 on virulence of C. posadasii, theinventors constructed Δcps1 strains using Agrobacterium mediated genereplacement. The deletion construct contained the hygromycin B gene fromEscherichia coli between a 1.1 kb CPS1 5′ flanking segment and a 1.2 kbCPS1 3′U flanking segment. The entire construct (5′flank-hph-3′flank)was cloned into pAM1145 between T-DNA border sequences at EcoRI site andtransformed into Agrobacterium tumefaciens strain AD965 and theresulting strain designated as A1594. The C. posadasii Δcps1 strainswere generated by co-cultivation of A1594 and C. posadasii germlings oninduction medium, followed by the selection of transformants on platedcontaining hygromycin. Forty-eight hygromycin resistant transformantswere picked and purified by successive re-streaking on plates containinghygromycin for three times. The CPS1 gene deletion was assessed by bothPCR and Southern hybridization. DNA was extracted from 24 hygromycinresistant strains and tested for deletion cassette using primers for hphgene and the CPS1 gene in an attempt to determine if the hygromycinresistant strains were homokaryons or heretokaryons. Based on theresults obtained from PCR, Southern hybridization was performed on theDNA of 17 transformants and wild type Silveira DNA restricted withEcoRI, using the hph gene, CPS1 gene, 5′flanking sequence and 3′flankingsequence amplicons as probes. When hybridized with the hph gene probe,strains 11, 19, 28 and 30, contained a 10.2 kb fragment indicative ofhomologous gene replacement event. All other transformants had varyingsized fragments indicating ectopic integration of the deletion cassette.The transformants were further tested with wild type CPS1 gene probe andthe putative gene replacement strains (11, 19, 28 and 30) lacked thewild type gene. Those which had a wild-type copy of the CPS1 gene had afragment of 5.1 kb, including the wild-type, Silveira.

To confirm the above result the inventors tested six transformants(namely 6, 11, 13, 19, 30 and 48) and wild type with 5′flankingsequences and 3′ flanking sequences as probes. All the homokaryotictransformants had a fragment of 10.2 kb with both the probes. Theseresults support the conclusion that at least four of the transformants(11, 13, 19 and 30) tested arose from a single homologous integrationevent of the CPS1 deletion cassette and other strains had anon-homologous integration resulting in ectopic transformants.

Example 5. Growth Rate and Spherule Size of CPS1 Mutants

The growth rate of the Δcps1 mutant was assessed at 24° C. and 37° C. onmedia containing with and without hygromycin as selectable marker.Except for minor differences, no major defects in growth rates wereobserved between the Δcps1 and wild type during the initial growthphase. At day 5, a slightly significant difference was observed betweenthe Δcps1 cultures grown on media containing with or without hygromycin(p=0.0474). However, by day 14, significant differences in growth ratesbetween the Δcps1 and wild-type were observed in cultures grown at 24°C., where the Δcps1 had a reduced growth when compared to the wild-type.This reduced growth in the mutant might be due to the accumulation ofsecreted metabolites in the media. In contrast, the growth rate wasalmost similar between the Δcps1 and wild-type at 37° C. and correlatedwith the lower accumulation of the putative metabolite.

To understand the effect of the CPS1 deletion on spherule size, theinventors inoculated induction media with arthroconidia and measured thesize of spherules at 24 hour intervals for a span of five days.Significant differences in spherule size were observed between the Δcps1and wild-type at all stages of spherulation (p<0.0001). On day five, themaximum size of Δcps1 spherules were 7.8 μM in diameter while thewild-type spherules measured 9.9 μM.

Example 6. Comparison of Virulence of Wild-Type and Δcps1 Strains

BALB/c mice were intranasally infected with 50, 211, 810 and 4400 viablearthroconidia of Δcps1 and with 59 arthroconidia of the strain Silveira,the wild-type parental strain, to assess the virulence of Δcps1 mutants(FIG. 1). Each concentration was tested in 12 different mice and themice were followed for 30 days post-infection. Mice challenged witharthroconidia derived from wild-type strain showed high rates ofmortality and nearly 30% of the animals died by day 15; all animals weredead by 19 days post-challenge. Interestingly, all animals infected witharthroconidia from Δcps1 mutant survived the length of the study (30days). The number of Δcps1 arthroconidia used for intranasal infectionshas no effect on the survival and the differences in the survival rateof mice between the wild-type and Δcps1 were significant.

The fungal burden in the mice infected with Δcps1 arthroconidia wasassessed by harvesting lungs and spleens at 30 days post-challenge. Fromthe 44 mice infected with the Δcps1 strain, only a single colony wasrecovered from the homogenate of the lung of one mouse infected with the810 dose. No CFU were recovered from the spleens of the mice. Testing ofthe single colony recovered from the 810-dose mouse determined that itwas a Δcps1 strain by PCR analysis.

Example 7. Δcps1 Strains Provide Protection Against Infection byWild-Type C. posadasii

To assess whether the avirulent Δcps1 strains are able to protect miceagainst subsequent infection by wild type Coccidioides, C57BL/6 micewere vaccinated, either intraperitoneally or subcutaneously, with 50,000Δcps1 arthroconidia, at two weeks boosted with the same number of sporesand after four weeks, infected with 90 arthroconidia of Silveira, astringent challenge as 50 spores is a lethal dose. The protectiveeffects of the Δcps1 strain was assessed two weeks after challenge. Bothlung weights and quantitative culturing of lungs revealed that the Δcps1strain provided significant protection against infection, with a 3 logreduction in fungal burden compared to the Ag2/PRA-CSA chimeric antigenand almost a 5 log reduction compared to the adjuvant alone controls(FIG. 2).

Example 8. Sensitivity to Hydrogen Peroxide

To test whether the CPS1 deletion has any effect on resistance tooxidative stress, the inventors examined the growth of Δcps1 atdifferent concentrations of hydrogen peroxide as a measure of oxidativestress. 2×GYE media was supplemented with different concentrations ofH₂O₂ (0 mM to 20 mM), inoculated with either Δcps1 or wild-type strainSilveira and growth assessed at either 24° C. or 37° C. Silveira was notable to resist more than 2 mM H₂O₂ whereas the Δcps1 was able to groweven at 8 mM H₂O₂. There were significant differences between the growthof Δcps1 and wild-type at both the temperatures (24° C. and 37° C.).Hygromycin in media had an effect on oxidative stress; the Δcps1 strainwas sensitive above 5 mM H₂O₂ when hygromycin was present in the mediawhile in the absence of hygromycin the mutant was able to grow on mediaup to 8 mM H₂O₂ at 37° C., or 6 mM H₂O₂ at 24° C. These resultscollectively indicate that the Δcps1 mutant was able to withstandoxidative stress at a higher level compared to the wild-type strain.

Example 9. Degenerate Spherule and Endospore Formation in an Avirulent

mutant strain of Coccidioides posadasii that induces protection in mice.

Animals: Female BALB/c mice were purchased from Harlan-Sprague-Dawley.NOD-SCID (NSG) mice were a generous gift from Jeffrey Frelinger (Dept.Immunobiology, The University of Arizona). Animals were housed andutilized according to NIH guidelines and all procedures were approved bythe University of Arizona Institutional Animal Care and Use Committee.NSG mice, which lack NK and all T- and B-cells and are severelyimmunodeficient, were housed under SPF (specific pathogen free)conditions until transfer into the ABSL3 laboratory for infection withCoccidioides.

Coccidioides strains: C. posadasii (Cp), strain Silveira, was grown tomaturity on glucose yeast extract agar plates at room temperature andarthroconidia were harvested by the spin bar method. This was used asthe virulent wildtype (WT) strain for challenge of vaccinated mice andas historical controls for histopathology slides to compare with Δcps1.The Δcps1 strain was transferred by plugs placed on fresh GYE plateswith hygromycin in the medium and grown to maturity (8 weeks).Arthroconidia were harvested as for WT.

Mouse Studies: For lung histopathology, NSG and BALB/c mice wereanesthetized with 80 mg/kg ketamine and 8 mg/kg xylazine IP and thetarget inoculum was instilled intranasally in 30 μl sterile saline forinjection. Mice were observed and weighed to monitor clinical condition.They were sacrificed using an overdose of inhalant anesthetic and lungsfixed in 10% buffered formalin or zinc acetate fixative. Serial or stepsections were cut and stained with H&E. Some studies were also stainedwith a Coccidioides-specific immunohistochemical stain.

For the protection study, 6-week old female BALB/c mice were vaccinatedintranasally or subcutaneously with 10,000 viable arthroconidia of Δcps1twice two weeks apart and challenged four weeks later with a target doseof 50 spores of strain Silveira. Control groups were vaccinatedsubcutaneously with MPL-SE adjuvant alone or 2 μg/mouse of chimericantigen with adjuvant, which is known to prevent death following lethalinfection in ≥90% of C57BL/6 mice. Mice were observed for 28 days andmoribund animals were euthanized as necessary throughout the study. Onday 28, surviving mice were sacrificed, and the lungs weighed, scored,and processed for quantitative determination of fungal burden. Colonieswere enumerated after 3 days' incubation at 37° C. Spleens were platedwhole on GYE plates and incubated up to one week at 37° C. to determineif disease disseminated.

Histopathology: For early studies, entire lungs of two mice per timepoint were cut in 5 μm sections and every fifth section was affixed toslides and stained with hematoxylin and eosin (H&E). For later studiesinvolving higher numbers of infectious arthroconidia, two serialsections were cut for every fifth step section and 10 steps total wereprepared on slides. One slide from each series was stained by H&E andone slide was immunohistochemically stained with a polyclonal goatanti-Ag2/PRA antibody that is specific for Coccidioides as previouslydescribed (Shubitz et al. Infection and Immunity 76:5553-5564, 2008).

Statistical Analysis: Lung fungal burdens were log transformed andcompared by ANOVA (Systat 10.0).

Results

NSG Mice Infected with 1030 Spores.

Six NSG mice were infected intranasally with 1030 spores of the Δcps1mutant strain. Mice were observed daily for lethargy, ruffled fur,inactivity, and dehydration. On day 6, two mice were sacrificed andlungs fixed in formalin. One mouse had occasional, pinpoint reddish-greylesions scattered throughout and the other was grossly normal.Examination of step sections (every 5^(th) section) through the entirelungs revealed no spherules. There were clusters in the alveolar spacesof large, foamy macrophages that appeared to correlate with the grosslyobservable lesions, but there were no fungal forms associated with theseareas. The remaining mice were observed until day 14 and all wereclinically normal at sacrifice with maintenance of body weight. Atnecropsy, two animals had occasional reddish-grey lesions (<12) asobserved in one of the mice at day 6 and the other two appeared normal.Two mice had lungs fixed in formalin and sectioned and the entire lungsof the other two were quantitatively cultured. One animal had no fungalgrowth and 8200 colony-forming units were recovered from the other.Again, no fungal organisms were observed in step sections through theentire lung of the two fixed mice, but the clusters of foamy macrophagesin the alveolar spaces were observed. However, the recovery of 8200colonies from one mouse suggests expansion of the infection in at leastone animal because that is an 8-fold increase over the target challenge.

NSG and BALB/c Mice Infected with 10,000 Spores.

Two NOD-SCID and two BALB/c mice were infected intranasally with 10,000spores of Δcps1 strain and one mouse of each strain was sacrificed onday 1 and day 3 post infection. Two serial sections were cut every fivesteps for a total of 10 steps (20 slides); one slide from each serialpair was stained with H&E and the other was stained specifically forCoccidioides. No organisms were seen with either stain on day 1 postinfection, but the Coccidioides-specific stain revealed low numbers ofvariably sized spherules on day 3 (FIGS. 9A and 9B). There were morespherules in the NOD-SCID mice than the BALB/c mice, but the pathologyin both strains was minimal. Spherules generally appeared thin walledand degenerating and were surrounded by clusters of neutrophils. This isin contrast to wildtype spherules of strain Silveira in mice at day 3,which appear to have thicker walls and are very round, often withdark-staining contents. Though abnormal spherule forms can be seen intissue due to cutting artifact, the degenerating condition of the Δcps1spherules appears to be constitutional. While day 3 spherules of strainSilveira appear to have one to a few layers of macrophages surroundingthem, the Δcps1 spherules are surrounded, and sometimes invaded, byneutrophils. (FIG. 9A and FIG. 9B). Neutrophils do not become asignificant cell type around normal spherules until rupture between day4 and 5 (FIG. 10). It has been previously reported that spherule ruptureand endospore release attracts neutrophils to the site. Whether thedegenerating condition of the walls of Δcps1 spherules is achemoattractant for neutrophils or whether the neutrophils have a rolein the condition of the spherules is currently unknown; however, FIGS.3A and 3B show that some spherules of Δcps1 appear abnormal without aneutrophilic infiltrate and it is possible that a defect in fungal wallstructure may be allowing substances that normally only attractneutrophils upon rupture to escape from the Δcps1 spherules duringearlier development. BALB/c mice (very susceptible, doses as low as 10spores IN lethal) were vaccinated IN and subcutaneously (SC) with Δcps1strain, challenged with 50 spores WT Coccidioides. First the mice wereinfected with 1030 spores, sacrificed on days 6 and 14 post-infectionfor histopathology. Histopathology from about 150 serial sections thatwere stained with H&E (routine stain) were negative for organisms. Noneof the mice became ill or died. For mice infected with 10,000 spores; nomice became ill and were sacrificed on days 1 and 3. Serial sectionswere stained with a Coccidioides-specific stain. Spherules werevisualized at Day 3 using the Coccidioides-specific stain in bothstrains of mice; more spherules were present in the in NSG mice; thespherules look degenerate (FIGS. 3A and 3B).

Upon initial scanning, spherules were not observed on the H&E stainedslides due to the paucity of organisms and the degenerating walls thateither take up stain poorly or do not stain at all. Because theinventors had serial sections to examine, the H&E stained slides werereviewed again after locating spherules with the Coccidioides-specificstain. Low power scanning of the slides revealed the sites of neutrophilaccumulation and examination of several of these sites at higher power(400-1000×) revealed remnants of spherule walls, or outlines of wallswith neutrophils both surrounding the original spherule and filling itin, leaving an outline where the wall existed or where fragments maystill be seen at high power (FIG. 10). One small, empty, thin-walledspherule was observed intact, but clearly is not undergoingendosporulation. (FIG. 10).

BALB/c Mice Infected with 25 Million Spores.

Mice were given an overwhelming dose of Δcps1 spores in order to makemore observations of the spherules in vivo and to determine the fate ofthe spherules over time since in all previous studies (n=3) where lowerdoses were used, the organism was entirely cleared and there was noresidual lung inflammation by day 14 post-infection. Eight BALB/c micewere given 25×10⁶ spores intranasally; two mice were sacrificed on day 1post-infection, one mouse each on day 3 and day 4, two mice on day 5,one mouse on day 7 and one mouse on day 10. Clinically, the mice beganto look lethargic and have ruffled fur on day 3 and two mice, instead ofone, were sacrificed on day 5 because they appeared ill enough thatmaintaining them longer would constitute unnecessary suffering. Theremaining two mice improved clinically after day 5 and appearedoutwardly normal at the time of sacrifice.

All mice had grossly visible lesions in the lung except for the micesacrificed at 24 hours. The lesions of mice on days 3-5 were diffuse,large, and pale and appeared edematous or “wet” rather than havingdiscrete 0.5-1.0 mm granulomas typical of early coccidioidal infectionin mice. The mice sacrificed on days 7 and 10 had grossly normal lungs.Five pairs of serial sections were made for each mouse and one of eachpair was stained with H&E and the other with the Coccidioides-specificstain. H&E stains revealed that the mice developed a severe suppurativepneumonia with edema by day 3. The beginnings of this are apparent onday 1, but it is diffuse by day 3. The neutrophil is the primaryinflammatory cell present. In addition, there are vast numbers ofdeveloping Δcps1 spherules in clusters in the middle of the suppurativeresponse. As with the sporadically visualized spherules from the miceinfected with 10,000 spores, they are highly variable in size and manyare degenerating, but some appear to be attempting to endosporulate.

On day 4, the largest proportion of the Δcps1 spherules have beeninvaded by neutrophils, but structures that appear to be endospores arereadily visible within spherules. They are highly variable in size andhave thin walls like the Δcps1 spherules themselves. WT spherulesrupture between day 4 and 5.

On day 5 the Δcps1 spherules appear similar to day 4, however, they aredistinct from WT in that the endospores have not been released or spreadout from the spherule as happens with normally rupturing sperhules.(FIG. 10). All of the endospores, even those where the walls of theparent spherule are degenerated, are completely surrounded byneutrophils which appear to be containing them in their originalposition. In terms of the general inflammatory response, by day 5 agranulomatous component is seen. Macrophages have moved into peripheralpositions surrounding the neutrophils and early granuloma formation ispresent. With wildtype spherules, the macrophages are present prior tothe arrival of the neutrophils in the early lesion.

By day 7 post-infection, the Δcps1 spherules appear to be decreased by≥90% compared to day 5 (visual estimate) and the inflammation ischaracterized by granulomas with a core of neutrophils and spherules.

By day 10, the infection is reduced to small, scattered granulomas withfewer than a dozen primarily empty spherules within (FIG. 12).

Protection of BALB/c Mice by Intranasal or Subcutaneous Vaccination withViable Δcps1.

The inventors have previously demonstrated that C57BL/6 mice can besignificantly protected against lethal Coccidioides infection byimmunizing with live spores of Δcps1. Because the inventors have beenable to show good protection of C57BL/6 mice but poorer protection ofBALB/c mice with recombinant vaccines in the past, and because in theseexperiments BALB/c mice are more susceptible to infection than C57BL/6mice, the inventors compared BALB/c mice vaccinated with Δcps1 versusthe Ag2/PRA-CSA chimeric recombinant vaccine that protects approximately90% of C57BL/6 mice against challenges of 50 spores.

BALB/c mice vaccinated with the recombinant antigen or adjuvant only alldied between days 13-15 post-infection, whereas all but one of the micevaccinated with Δcps1 survived until day 28 (P<0.001) (FIG. 6). Therewas no statistical difference between the route of vaccination with theΔcps1. Lung fungal burdens were quantified for surviving mice on day 28and even though the intranasally vaccinated mice had lower fungalburdens (FIG. 7), there was no statistical difference between the groups(P=0.241).

Example 10. Virulence of Mutant Strains Compared to Wild Type Silveirain Susceptible Mice

8 week old C57BL/6 female mice were infected with target doses as listedin Table 2 with spores counted in the lungs.

TABLE 2 Type and Amount of Spores Inoculated and Present in Lungs.Spores Number of Spores counted Group Fungus inoculated mice in lungs 1WT 50 12 59 2 Δcps1 50 12 51 3 Δcps1 250 12 211 4 Δcps1 1000 12 810 5ΔLOM 50 12 51 6 ΔLOM 250 12 310 7 ΔLOM 1000 12 900

Mice in the L-ornithine monooxygenase mutant (ΔLOM) 1000 spore and 250spore groups started to become moribund on day 9 and day 10,respectively. All of the 1000 spore mice were euthanized on day 9 andthe 250 spore mice were euthanized on days 10 and 11. Disease scoreswere high in all animals, except a single mouse (#10) in ΔLOM 1000group. The animal lungs were positive for growth, but likely this mousereceived only a fraction of the target dose. Wild type mice (59 spores)became moribund on days 13-19, and the ΔLOM 50 mice on days 13-16. Thesetwo groups appeared clinically similar and had similar disease scores atnecropsy.

The Δcps1 infected mice remained healthy throughout the observationperiod of 4 weeks. Though the original plan was to quantitate the fungalburden at the end of the study with any remaining mice, a modified planwas made to plate entire lungs and spleens if animals had no grossevidence of disease at necropsy. None of the mice had observable lesionsat day 28 post-infection, and none of the slides from day 11post-infection showed any pockets of spherules or any inflammation, noteven perivascular/peribronchial infiltrates. Tissues were incubated onGYE agar plates for 9 days.

One mouse (#9, 1000 Δcps1 spore group) had growth in the lung but notthe spleen. The strain that grew from this lung was saved for analysis.Statistical analysis of the survival curves was performed using aKruskal-Wallis. 1) ΔLOM at each dose was statistically different fromthe other doses (p<0.001). 2) ΔLOM 50 sp was significantly differentfrom WT 59 spores (p<0.001), with the mutant mice having earlier deathsthan the WT mice. This occurred with a Δstel2 C. posadasii mutant aswell. Histologically, no differences were seen between the WT mice andΔLOM mice on HE stained slides. 3) ΔLOM at the higher doses was alsosignificantly different from WT 59 sp. 4) None of the Δcps1 groups wasdifferent from each other (p=1.0), and all were different from the otherstrains.

Histopathology: The ΔLOM mice did not appear different from the WT micewhen inoculated with a similar dose (˜50 spores). The ΔLOM miceinoculated with higher doses had enormous numbers ofspherules/endospores in all fields of the lungs. Δcps1 infected mice hadno evidence of infection or inflammation in the lung sections examined.Though entire lungs were submitted in 10% formalin, only a single slidewith a single slice of tissue was reviewed.

Fungal burden: Nothing was quantitated in this study because of themoribund condition of 4 groups prior to the scheduled date of sacrificeand the lack of gross lesions in the remaining 3 groups of mice. Formoribund mice, disease scores were 3-4 L with 6 mice also having visiblelesions on the spleen. Growth was present in both lungs and spleen ofmost mice (with the exception of 2 ΔLOM 1000 mice which had no growth inspleen). For the healthy-appearing Δcps1 infected mice, plating ofentire lungs and spleens yielded growth from the lung of a single mouseinoculated with 810 spores. This confirms that the mutant strain canreproduce in the host, but that the virulence of the strain is greatlydiminished.

Example 11. Evaluation of a RYP1 Knockout in Coccidioides

The RYP1 gene (Nguyen and Sil, 2008, Proceedings of the National Academyof Sciences, USA 105:4880-4885) was knocked out in Coccidioides strainSilveira. Deletion of the RYP1 gene leads to avirulence in Histoplasmacapsulatum because the mold cannot switch to the pathogenic yeast phase.In Coccidioides, deletion of the RYP1 gene (CIMG_02671.3) appears toinhibit the change to the spherule/endospore phase in vitro and istherefore anticipated to be entirely avirulent. This strain was comparedwith high doses of Δcps1.

8 week old C57BL/6 mice were challenged with Δryp1 spores, Δcps1 sporesand WT spores in doses shown in Table 3, with the post infection platecount.

TABLE 3 Infection of Mice with Δryp1, Δcps1 and WT Spores. Type ofAmount of Number of Post infection spores spores mice plate count WT 5012 53 Δcps1 1000 12 4400 Δryp1 50 12 47 Δryp1 1000 12 994

Mice infected with the WT strain began to become moribund on day 12post-infection. The largest proportion were moribund on day 14. By day19, all WT mice were dead except one animal that demonstrated weightgain and was suspected to be uninfected. The Δcps1 and Δryp1 inoculatedmice remained clinically normal. The #11 and #12 mice from each groupwere sacrificed for histopathology on day 14. Mice challenged with Δcps1and both doses of Δryp1 had no weight loss and no observable lunglesions. The #11 WT mouse was also negative for lung lesions and had noweight loss, but #12 was as expected. At sacrifice at 28 days, theliving WT mouse had one medium sized granuloma in the lung and twosplenic granulomas. All the mutant Δcps1 and Δryp1 inoculated mice weregrossly negative. Organs were plated in toto on GYE plates.

All the organs plated except from the WT mouse were negative, bothspleens and lungs, after 8 days of incubation. Histopathology of organsof the Δcps1 and Δryp1 infected mice 14 days post-infection showed noorganisms in any of the mice. A couple of animals had mild, focal,inflammatory lesions, only 1 per mouse. No organisms were seen. WT #11had no histologic evidence of disease, while #12 had a score of 4/5 forlungs, plus abscesses that consist almost exclusively of neutrophils andspherules/endospores in the spleen. Average days survived and averageinfection and sacrifice weights are provided in Table 5.

The Δcps1 strain appears in this corroborative study to benonpathogenic, and the Δryp1 strain was also nonpathogenic, as expectedbased on work done with the ortholog of this gene in Histoplasmacapsulatum (Nguyen and Sil, 2008, Proceedings of the National Academy ofSciences, USA 105:4880-4885). The survival of two WT mice was unusualand likely due to technical issues, such as the mouse swallowing thedose instead of inhaling it. It is seldom observed that C57BL16 surviveto 4 weeks if infected, but this animal did have systemic disease asevidenced by growth of Coccidioides from the spleen as well as lungs.

TABLE 5 Survival and Weights of Mice Used in Study. Avg. Day Avg.Infection Avg. Sacrifice Mouse # survived weights Weights Δryp1 50 24.919.2 21.3 Δryp1 100 24.9 19.8 22.0 Δcps1 5000 24.9 19.9 22.1 WT 50 15.519.3 15.2

Example 13. Evaluation of Immunity Provided by Avirulent CoccidioidesMutants Following Inoculation

A study was performed to determine if two of the avirulent Coccidioidesmutants will provide immunity to mice as a vaccine. Δryp1 and Δcps1knockouts are the most interesting of the avirulent strains studied.Ryp1 is required to form spherules/endospores as the lack of the geneprevents the fungus from undergoing the transformation. Cps1 is involvedin small molecule synthesis, and the gene product is required forpathogenicity.

6 week old, female, C57BL/6 mice were inoculated with Δryp1 spores,Δcps1 spores, Chimeric Ag2/PRA-CSA antigen only (Chim Ag), HSPvar only(HSF), and Adjuvant plus Sac Supe (Sac Supe) with the amount andinjection method shown in Table 6. SC=subcutaneously.IP=intraperitoneal. Average lung weights are also shown in Table 6.

TABLE 6 Effects of Δryp1 and Δcps1 Inoculation on Lung Weights. SporesNumber of Avg Lung Group inoculated IP/SC Mice weight (g) Δcps1 50000 IP8 0.3 Δcps1 50000 SC 8 0.3 Δryp1 50000 IP 8 0.79 Δryp1 50000 SC 8 0.8HSF 2 μg SC 8 0.6 Chim Ag 2 μg SC 8 0.4 Sac Supe 2 μg SC 8 0.7

Mice receiving the mutant strains were given 5000 viable arthroconidiaof the respective strain intraperitoneally or subcutaneously. Allanimals were boosted at 2 weeks, then rested for 4 weeks before beingchallenged intranasally with 50 arthroconidia of C. posadasii WT strainSilveira. Mice were sacrificed two weeks after challenge. Peritoneallesions and subcutaneous injection sites had culture and histopathologyperformed at necropsy.

Mice were challenged intranasally with wild type strain Silveira 4 weeksafter boosting. Post-infection plate counts revealed that the micereceived 90 spores. The 90 spores is a pretty stringent challenge.

Mice had weight loss in groups 3, 4, 5, and 7 by day 10 post-infection.With fluid support, most mice survived. Two mice died. Disease scoreswere recorded, but this time lung weights were captured as a better formof data to subject to statistical analysis. Disease scores and lungweights correlated pretty well, but the lung weights are not subjectiveand don't depend on separating a continuum of disease, especially inmice that are borderline between 2 and 3 and 3 and 4 disease scores.Almost all mice injected with Δcps1 mutant strain had visible granulomasin the subcutaneous tissues or 1-2 mm white lesions in the omentum.These were collected for culture.

The injection of the Δcps1 mutant strain either IP or SC resulted in thelowest lung weights in this experiment. All animals had evidence of agranulomatous lesion at the injection site, indicating that the mutantstrain may have reproduced locally, providing sufficient antigen toresult in a memory response that led to protection. A few IP injectedanimals also had 1-2 mm granulomas in the omentum. The Δryp1 strainappeared to provide no protection to the mice. This could be because itundergoes no growth at all in the host and the 25,000 arthroconidia arenot sufficient to induce a memory response in the mice.

Quantitative Lung Cultures: Box plot of the CFU from the seven groups inthe study. It is clear that the Δcps1 mutant offered the greatestprotection with a 2 log lower average lung weight compared to thepositive control chimeric antigen group (FIG. 14). None of the othervaccine groups showed protection compared to control and the statisticalanalysis of both lung weights and total lung CFU bear this out.

Chimeric antigen, the “gold standard” resulted in 2 logs higher lungfungal burden than the Δcps1 strain but had significant reduction (˜3logs) in fungal burden compared to controls. Mice looked prettycomparable to those in other studies with a similar challenge (˜100spores). While this is the positive control antigen in the studies, itwas known that there is room for a better antigen and this study againreinforces that.

What is claimed is:
 1. An immunogenic vaccine composition, comprising:an avirulent Coccidioides arthroconidia comprising a geneticallymodified or missing cyclic peptide synthase (CPS1) protein; and apharmaceutically acceptable carrier.
 2. The immunogenic vaccinecomposition of claim 1, wherein the composition is formulated for humanadministration.
 3. The immunogenic vaccine composition of claim 1,wherein the vaccine composition is formulated for intranasaladministration.
 4. The immunogenic vaccine composition of claim 1,wherein the vaccine composition is formulated for subcutaneousadministration.
 5. The immunogenic vaccine composition of claim 1,wherein the vaccine composition is formulated for intramuscularadministration.
 6. The immunogenic vaccine composition of claim 1,wherein the CPS1 protein is genetically modified or missing by deletionof at least a portion of the CPS1 gene encoding the CPS1 protein.
 7. Theimmunogenic vaccine composition of claim 1, wherein the CPS1 protein isgenetically modified or missing by deletion of the DNAmethyltransferase-associated protein (DMAP) binding domain region of theCPS1 gene encoding the CPS1 protein.
 8. The immunogenic vaccinecomposition of claim 1, wherein the CPS1 protein is genetically modifiedor missing by deletion of the adenosine monophosphate (AMP) bindingdomain region of the CPS1 gene encoding the CPS1 protein.
 9. Theimmunogenic vaccine composition of claim 1, wherein the CPS1 protein isgenetically modified or missing as a result of a deletion of at leastone regulatory element of the CPS1 gene encoding the CPS1 protein. 10.The immunogenic vaccine composition of claim 1, wherein the CPS1 proteinis genetically modified or missing as a result of a deletion of at least10% to at least 90% of the CPS1 gene encoding the CPS1 protein.
 11. Theimmunogenic vaccine composition of claim 1, wherein the CPS1 protein isgenetically modified or missing as a result of at least one nucleotidesubstitution in the CPS1 gene encoding the CPS1 protein.
 12. Theimmunogenic vaccine composition of claim 1, wherein the CPS1 protein isgenetically modified or missing as a result of at least one insertion inthe CPS1 gene encoding the CPS1 protein.
 13. The immunogenic vaccinecomposition of claim 1, wherein the CPS1 protein is genetically modifiedor missing as a result of at least one interruption in the CPS1 geneencoding the CPS1 protein.
 14. The immunogenic vaccine composition ofclaim 1, wherein the Coccidioides arthroconidia are Coccidioidesposadasii arthroconidia or Coccidioides immitis arthroconidia.
 15. Amethod of protecting a mammal against infection by coccidioidomycosis,comprising administering the immunogenic vaccine composition of claim 1to the mammal.
 16. The method of claim 15, wherein the mammal is ahuman.
 17. The method of claim 16, wherein administering the vaccinecomposition comprises intranasally administering the vaccine compositionto the human.
 18. The method of claim 16, wherein administering thevaccine composition comprises subcutaneously administering the vaccinecomposition to the human.
 19. T The method of claim 16, whereinadministering the vaccine composition comprises intramuscularlyadministering the vaccine composition to the human.
 20. The method ofclaim 16, wherein administering the vaccine composition comprisesadministering the vaccine composition by a syringe, an inhaler or anebulizer to the human.
 21. The method of claim 15, whereinadministering the immunogenic vaccine composition comprisesadministering a first dose of the immunogenic composition followed by asecond dose of the immunogenic composition within three weeks of thefirst dose.
 22. The method of claim 15, wherein administering theimmunogenic vaccine composition comprises administering a first dose ofthe immunogenic composition followed by a second dose of the immunogeniccomposition within two weeks of the first dose.
 23. The method of claim15, wherein administering the immunogenic vaccine composition comprisesadministering a first dose of the immunogenic composition followed by asecond dose of the immunogenic composition within two to 4 weeks of thefirst dose.
 24. The method of claim 15, wherein administering theimmunogenic vaccine composition comprises administering a first dose ofthe immunogenic composition followed by a second dose of the immunogeniccomposition within 2-4 weeks of the first dose and then a single doseadministered annually thereafter.